Autologous bone marrow derived mesenchymal stem cells are safe for the treatment of Achilles tendinopathy

Achilles tendinopathy is a disabling condition that affects more than 50% of runners. Pre-clinical studies in a large animal model of naturally-occurring tendinopathy similar to human Achilles tendinopathy has shown benefits of autologous bone marrow-derived mesenchymal stem cell (MSC) implantation. However, MSCs are advanced therapies medicinal products (ATMPs), with strict regulatory requirements. Guided by the regulator we carried out a first in man study to assess the safety and efficacy of autologous MSC injection in human patients with non-insertional Achilles tendinopathy. Ten patients, mean age 47 with mid-portion Achilles tendon pain and swelling for more than 6 months, underwent autologous cultured cell injections (median 12.2 × 106, range 5–19 × 106 cells) into their Achilles tendon. At 24 weeks follow-up, no serious adverse reactions or important medical events were observed. MOXFQ, EQ-5D-5L, and VISA-A scores improved clinically at 12 and 24 weeks. VAS pain improved increasingly at 6, 12 and 24 weeks. MOXFQ Pain and VISA-A Scores improved > 12 points from baseline to 24 weeks in 8 patients. Maximum anteroposterior tendon thickness as measured by greyscale US decreased by mean 0.8 mm at 24 weeks. This phase IIa study demonstrated the safety of autologous MSC injection for non-insertional Achilles tendinopathy and provides proof-of-concept of the technique in patients, all of whom had previously failed conservative treatments for chronic disease and leads the way for a larger randomised controlled trial.


Clinical outcome measures
Patient-reported clinical outcome measures at baseline and 6, 12 and 24 weeks post-injection are summarized in Table 1.All three MOXFQ subscales, where a lower score represents a better outcome, improved significantly at 6, 12 and 24 weeks versus baseline, aside from the social interaction subscale from baseline to 6 weeks.The MOXFQ pain score improved from mean (± sd) 52 ± 11 at baseline to 24 ± 18 by 24 weeks (p < 0.001).The change in MOXFQ pain score exceeded the minimal clinically important difference (MCID) of 12 points in 8 of 10 patients.
EQ-5D-5L and EQ-VAS scores improved significantly from baseline to each subsequent timepoint.At 24 weeks, EQ-5D-5L scores increased by median 0.16 units versus baseline, while EQ-VAS scores increased by mean 14 units.Similar improvements were observed in VISA-A scores, which increased from baseline with greater differences at each subsequent time point, and mean increase of 22 units at 24 weeks (p < 0.001).VAS pain scores were significantly lower at 12 and 24 weeks versus baseline, with mean reduction of 23 units (p = 0.03) at 24 weeks.The SAS increased from baseline to 24 weeks by mean 1.6 units (p = 0.02).One patient refused to attend the final review as he informed the trial manager, he felt he was "cured" and back to running.Although all the other 9 patients completed questionnaires at 24 weeks, not all questions were answered.

Successful outcomes
At 24 weeks, 8 of 10 patients (80%; 95% CI 44% to 97%) had a successful outcome, defined as changes in outcome scores from baseline meeting or surpassing the MCID (i.e., > 12 points on the MOXFQ Pain Subscale and VISA-A).

Ultrasound outcomes
Ultrasound outcomes were determined separately by each radiologist.The maximum AP thickness of the tendon did not vary significantly from baseline through 12 weeks (Table 2), but decreased by a mean of 0.8 mm at 24 weeks (p = 0.02).Distance of the lesion from the calcaneum dorsal cortex, size of focal change, and percentage of disorganised tissue (i.e., type of fibre) did not vary significantly from baseline through 24 weeks.Furthermore, there was no change in the Doppler assessment of neovascularization over 24 weeks.UTC was only obtained on 6 of the 10 patients at 24 weeks due to logistical challenges but no significant changes in fibre type was observed (Fig. 4).

Discussion
This phase IIa, proof-of-concept, single-arm study determined the safety of autologous BMMSC injection for AT in a clinical setting.No serious adverse reactions or important medical events were observed in all 10 patients.In these patients, who had previously failed conservative treatments for chronic AT, treatment with autologous BMMSC injection provided relief of symptoms and notable improvement in MOXFQ scores, EQ-5D-5L and EQ-VAS scores, and walking improved through 24 weeks post-injection.
Existing treatments relying on inflammation-repair sequences have shown equivocal results 5,21,22 and so a regenerative solution is conceptually attractive.The mechanism of action of MSCs is not fully understood.It may be tropic, such that MSCs secrete paracrine factors to coordinate the body's response, including an immunomodulatory role 23 , or it may be the direct action of stem cells becoming tendon cells 13 .We do not know if the observed benefit is due to direct effects, interactions relating to the MSCs, or a placebo effect, as this study was not controlled.
This study used autologous cells.Ilic and Atkinson 15 investigated the injection of allogenic MSCs derived from human placenta into the Achilles tendon in six patients was safe, with no adverse reactions four weeks following injection, but they did not evaluate clinical outcomes.
Ultrasound tissue characterization (UTC) is a reliable imaging method for clinical assessment of AT 24 .The dynamics of grey levels, using histomorphology of the tissue specimen as a reference, is strongly related to tendon    www.nature.com/scientificreports/ The Achilles tendon was visualised tomographically with ultrasound tissue characterization (UTC; UTC Imaging, Stein, Netherlands) 28 .A high-resolution linear ultrasound probe (7-12 MHz, Teratech, USA) fixed to a tracking device moved along the tendon to collect adjoining transverse ultrasound images every 0.2mm over 12cm.The patient was positioned prone on the examination table with the ankle caudal to the edge.To achieve maximum passive dorsiflexion, the examiner pushed their knee against the patient's forefoot.All scans were performed by one experienced examiner (LM), distally to proximally 34 .Images were captured, stored, and compounded to create a three-dimensional (3D) volume block for tomographical reconstructions (i.e., transverse, sagittal, coronal, and 3D coronal planes).Validated UTC algorithms (UTC 2010, UTC Imaging) discriminated four distinct echotypes of matrix integrity: Type 1: aligned fibrillar structure; Type 2: wavy fibres; Type 3: haphazard aligned fibres; and Type 4: amorphous material with no fibres 34 .For analysis, Types 1 and 2 represented organized tissue, and Types 3 and 4 represented disorganized tissue 28 .The percentage of each echotype was measured within the BMMSC injection region.

Treatment procedure
The patient was placed in a left lateral decubitus position.Approximately 8mL of bone marrow aspirate was collected from the right posterior superior iliac crest into a heparinised syringe under local anesthetic and sent to a GMP facility licensed by the UK MHRA for production of human stem cells as investigational medicines.The patient was monitored after the procedure for adverse events and prescribed pain medication as indicated.Any adverse events during or immediately following bone marrow harvest were recorded.
Bone marrow-derived mesenchymal stem cells (BMMSCs) were isolated from bone marrow mononuclear cells by plastic adherence for 16-24 h using a standardized, GMP-compliant protocol of Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum.The adherent cells were expanded through up to three passages over 28-43 days to obtain 4-20 million BMMSCs.A dose of 4 × 10 6 cells was considered the minimum required for efficacy 12,13 , and the ideal dose achieved was 20 × 10 6 cells.The actual dose varied between these values (median 12.2 × 10 6 , range 5 to 19 × 10 6 ), dependant on culture conditions.The final, aseptic products were > 90% MSC (CD73 + /CD90 + /CD105 +), > 95% viable, and formulated as investigational medicinal products in suspension in two syringes ready for direct injection.The syringes were shipped by courier to the clinical site on the day of treatment.
Stem cell injection was performed approximately 5 (range: 4-6) weeks after bone marrow harvest in an outpatient setting.A suspension of purified BMMSCs in 1 mL of Dulbecco's Modified Eagle Medium was injected percutaneously under local anaesthetic using in-plane ultrasound guidance by an experienced, trained radiologist longitudinally along the length of the tendon.The area of maximal anteroposterior tendon thickness was identified, and the needle inserted 1 cm proximal to the lesion and passed across the abnormal tendon, ending 1 cm distal (Fig. 3).The injection was administered as the radiologist pulled the needle backwards through the abnormal part of the tendon.An adhesive, sterile, dry dressing was applied.Patients were monitored for at least 2 h following implantation for immediate adverse effects.Two days after the procedure, the trial coordinator telephoned all patients to check their well-being and record any adverse events.
Patients underwent a standard course of physiotherapy for Achilles tendinopathy, comprised of a 12-week eccentric loading programme 35 .

Outcome assessments
Patient demographics, comorbidities and concomitant medications were recorded at baseline.Patients completed several outcome measures at baseline that represented the best methods we could identify to assess foot and ankle pathology and Achilles tendinopathy 36 : Victorian Institute of Sports Assessment Achilles (VISA-A) Questionnaire 37 , Visual Analogue Score (VAS) for pain (0-100 scale) 38 , Manchester Oxford Foot and Ankle Questionnaire (MOXFQ) 39,40 , EuroQol 5-dimensions 5-levels (EQ-5D-5L) 41 , and Stanmore Sporting Activity Scale (SAS) modified from the UCLA sports activity score 42 .
At 6, 12 and 24 weeks following MSC implantation, patients underwent clinical assessment, adverse event review, conventional ultrasound imaging, UTC, and completed the VISA-A, VAS pain, MOXFQ, and EQ-5D-5L.The SAS was completed at 24 weeks.
The primary outcome for this study was the rate of serious adverse reactions.Secondary outcomes included efficacy as determined by the VISA-A, VAS Pain, MOXFQ, EQ-5D-5L, EQ-VAS, and SAS, and overall "success at 24 weeks, " defined as all of these: (i) reduction of ≥ 20 points in VAS pain 43 ; (ii) improvement in SAS score; and (iii) increase of > 12 points in VISA-A score (i.e., the minimal clinically important difference, MCID) 44 .Ultrasound outcomes included maximum anteroposterior tendon thickness, lesion distance from the calcaneum, level of neovascularisation, and UTC fibre type (Fig. 4).

Statistical analysis
Continuous variables are reported as means and standard deviations if normally distributed and as medians and interquartile (IQ) ranges if not normally distributed.Categorical variables are reported as frequencies and percentages.The primary safety endpoint and the secondary endpoint of overall success were calculated with exact binomial confidence intervals.
Exploratory analyses were performed to examine changes in secondary outcomes from baseline to postoperative time points, using a paired t-test (normal distribution) or Wilcoxon matched-pairs test (not normally distributed).All variables were continuous.
Inter-rater reliability of conventional ultrasound measurements was evaluated using the intra-class correlation (ICC) for continuous measurements and the weighted kappa statistic for ordinal measurements.Data from all four time points were included in each analysis.

Figure 2 .
Figure 2. (a) Transverse greyscale ultrasound image of an enlarged Achilles tendon, with low reflectivity on its superficial aspect affecting approximately 50% of the anteroposterior diameter of the tendon.(b) Longitudinal ultrasound image with colour Doppler.The tendon is fusiform in morphology, consistent with chronic noninsertional tendinosis, and the superficial aspect of the tendon shows low reflectivity.Moderate (grade 3) vascularity is seen in the tendon.

Figure 3 .
Figure 3. Stem cell injection performed under local anesthetic and greyscale ultrasound control: (a) intraoperative image; (b) longitudinal ultrasound image.The needle was inserted from distal to proximal and lies within the superficial low reflectivity region of the thickened tendon.Injection was performed while withdrawing the needle.

Figure 4 .
Figure 4. Longitudinal greyscale ultrasound of the right Achilles tendon (A) before injection-cursors measuring focal low reflectivity tendinosis); (B) similar image showing needle positioning; and (C) longitudinal colour doppler mode on grey scale ultrasound six months post injection showing improvement in tendon swelling and improved reflectivity of the tendon.

Table 1 .
Clinical Outcome Measure Scores shown as mean ± standard deviation, and change from baseline shown as mean with 95% confidence interval in brackets.n/a not applicable.^ Reported as mean change with 95% confidence intervals.*Reported as median [inter-quartile range] at each time point, with median change [95% CI].

Table 2 .
Ultrasound outcomes, shown individually for each radiologist (Rad), and UTC outcomes, reported as mean ± standard deviation, unless noted otherwise.Rad radiologist, Max AP Thickness maximum anteroposterior thickness, UTC Ultrasound tissue characterization.*Reported as median [inter-quartile range] at each time point, along with median change [95% CI].^ Disorganized = Type 3 (i.e.haphazard fibres aligned) + Type 4 (i.e.amorphic material or no fibres.